Journal
SCIENTIFIC REPORTS
Volume 7, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/srep42716
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Funding
- Grant for Adaptable and Seamless Technology Transfer Program through Target-driven R D [AS2525029M]
- Japan Science and Technology Agency (JST)
- Basic Science and Platform Technology Program for Innovative Biological Medicine
- Japan Agency for Medical Research and Development (AMED)
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We have attained a chemically modified DNA aptamer against salivary alpha-amylase (sAA), which attracts researchers' attention as a useful biomarker for assessing human psychobiological and social behavioural processes, although high affinity aptamers have not been isolated from a random natural DNA library to date. For the selection, we used the base-appended base (BAB) modification, that is, a modified-base DNA library containing (E)-5-(2-(N-(2-(N6-adeninyl) ethyl)) carbamylvinyl)-uracil in place of thymine. After eight rounds of selection, a 75 mer aptamer, AMYm1, which binds to sAA with extremely high affinity (K-d < 1 nM), was isolated. Furthermore, we have successfully determined the 36-mer minimum fragment, AMYm1-3, which retains target binding activity comparable to the full-length AMYm1, by surface plasmon resonance assays. Nuclear magnetic resonance spectral analysis indicated that the minimum fragment forms a specific stable conformation, whereas the predicted secondary structures were suggested to be disordered forms. Thus, DNA libraries with BAB-modifications can achieve more diverse conformations for fitness to various targets compared with natural DNA libraries, which is an important advantage for aptamer development. Furthermore, using AMYm1, a capillary gel electrophoresis assay and lateral flow assay with human saliva were conducted, and its feasibility was demonstrated.
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